Alzheimer's disease (AD) is a neurodegenerative disease characterized by the deposition of amyloid plaque in the extracellular space, formation of neurofibrillary tangles in neurons, and extensive neuronal loss. Growing evidence has shown that increased beta amyloid (A) peptide accumulation, aggregation, and deposition in the brain are central to the pathogenesis of AD (1). Recently, the advent of methodologies to reprogram fully differentiated tissues, such as skin, into human-induced pluripotent stem cells (hiPSCs) has made it possible to study AD in a human physiological context. Cell-based human models for AD will be created by directly reprogramming skin fibroblasts from AD patients and controls into hiPSCs. This proposal will focus on reprogrammming skin fibroblasts from sporadic AD patients containing two copies of the 4 allele of the apolipoprotein-E (APOE) gene and control subjects containing two copies of the APOE 3 allele. Previously established hiPSC lines derived from familial AD patients with mutations in the amyloid precusor proteins (APP) and preselinin (PS) genes will be obtained and differentiated into neuronal cells, including cholinergic neurons. There are three primary aims for this R21 proposal. In Aim 1, fibroblasts from AD patients carrying the APOE 4 allele and controls carrying the APOE 3allele will be reprogrammed into hiPSCs and then differentiated into neurons. Several parameters associated with AD development such as A expression level, A fibrilgenesis, neuroinflammation, synapse formation and function will be analyzed at different timepoints after neuronal differentiation. Thi will illustrate whether hiPSC-derived neurons can recapitulate the disease progression of AD in vitro. In Aim 2, A 1-42 peptide will be used to treat the hiPSC-derived neurons at low concentrations resembling in vivo A concentration and at high concentrations that induce neuronal toxicity. Various indicators for neuroinflammation, neuronal degeneration and cell death will be measured to examine whether hiPSCs-derived neurons carrying the APOE 4 allele react differently to A treatments compared to those carrying the APOE 3 allele. In Aim 3, gene expression profiles of hiPSC-derived neurons will be generated using gene expression microarrays. Genes with altered expression in familial AD patients or patients with the APOE 4 allele will be cross-referenced with a list of genes modulating A neurotoxicty that we have generated through a genome-wide shRNA library screen. Overlapping genes of interest will be further studied using lentiviral overexpression or shRNA-mediated repression in hiPSC-derived neurons. Studies of hiPSC-derived neurons from AD patients carrying the APOE 4 allele have the potential to significantly advance our understanding of the complex mechanisms underlying APOE 4-dependent increase of the risk for AD. Results from the proposed studies will likely lead to development of hiPSC-derived neurons-based assays for screening genes or compounds that lead to potential new drug therapies for the treatment of AD. PUBLIC HEALTH RELEVANCE: The goal of this proposal is to create human cell-based models for Alzheimer's disease (AD) by reprogramming skin samples from sporadic AD patients with two copies of the APOE 4 allele into human induced pluripotent stem cells (hiPSCs). By differentiating these disease-specific hiPSCs into neurons, we will investigate the underlying mechanisms by which the apoE4 isoform mediates increased risk for AD. Ultimately, we aim to develop genetic or chemical treatments that disrupt molecular pathways that lead to AD.